Recent advances in the development and application of molecularly targeted therapies for cancer have generated promising new treatments. However, a major obstacle to achieving disease-free survival is drug resistance. The long-term objective of this research is to investigate the mechanisms of drug resistance and target resistant tumors for treatment. This application will specifically determine the role of EphA2 receptor tyrosine kinase in intrinsic and acquired resistant to EGFR/HER2 inhibitors. EGFR and HER2 belong to the ErbB family of receptor tyrosine kinases (RTKs) that are critical for tumor initiation and metastatic progression. Several therapeutic strategies for targeting EGFR and HER2/ErbB2 have been developed, including kinase inhibitors such as Iressa (Gefitinib), and antibodies such as herceptin (Trastuzumab). In spite of initial efficacy, intrinsic and acquired drug resistance is frequently observed in the clinic. We found that one of the mechanisms of resistance to anti-EGFR/HER2 therapy is up-regulation of EphA2, a member of Eph family RTKs. EphA2 has been linked to many types of cancer, including NSCLC, glioblastoma, melanoma, breast, colorectal, bladder, prostate and ovarian carcinomas. Moreover, the level of EphA2 expression correlates with tumor malignancy and patient survival. We recently reported that EphA2-deficiency impairs tumor initiation and metastatic progression in tumors over-expressing the ErbB2 oncogene. Biochemical analyses revealed that EphA2 forms a complex with HER2 or EGFR and promotes activation of MAPK and Rho GTPase. Additionally, MMTV-Neu tumors are sensitive to therapeutic treatment of EphA2, suggesting that EphA2 plays an important role in HER2/EGFR-dependent tumor progression. Based on these data, we hypothesize that overexpression of EphA2 receptor confers intrinsic and/or acquired resistance to anti-HER2/EGFR treatment. To test this hypothesis, Specific Aim 1 will investigate the role of EphA2 in intrinsic and acquired resistance to EGFR/HER2 inhibitors, using a novel anti-EphA2 human monoclonal antibody. Specific Aim 2 will determine the molecular mechanisms through which EphA2 is activated in resistant tumor cells. Together, these studies will make significant advances towards the understanding the mechanism of drug resistance to Her2/Erb2 based therapies. Success of this project will provide a strong rationale for rapid clinical development of anti-EphA2 therapeutics for treatment of EGFR/HER2 resistant tumors, which will greatly benefit the Veteran population.